In this renewal, we enter a third phase of our long term goal to understand the diverse reactivity of heme enzymes and to use that information to generate engineered forms with novel catalytic properties. The primary hypothesis that has driven these studies is that the chemical reactivity displayed by heme enzymes resides partially, but not exclusively, within the heme cofactor, and a significant role of the protein is to limit or direct access of substrates to this reactive center in ways that result in specific catalysis. Clearly, this hypothesis falls short, for many examples exist where the protein directly modulates the activity of the heme. It has thus been our goal to delineate the boundaries between these two roles for the protein, and then use this information to introduce new sites where novel substrates interact with the heme cofactor, take advantage of existing heme-protein interactions, and induce novel catalytic reactions. The first phase of this project was characterized by its emphasis on the physical, spectroscopic and functional properties of heme enzymes, primarily cytochrome c peroxidase (CCP) as a model. This work has given support to unifying themes that suggest how reactions of some heme enzymes might be recruited into the scaffold of others. In the second phase, we have developed an approach to introduce small-molecule binding sites into the heme environment by "cavity complementation", the creation of binding sites by mutagenic deletion of amino acid side-chains. This approach has provided new ways to test ideas about the diversity of heme enzyme function. It has also come up against some of the limitations of rational engineering to generate enzymes with tailor-made functionality. In the next period, we seek to begin development of an experimental method for the directed evolution of heme proteins and enzymes that bind compounds of the investigator's choice. The evolved proteins will be based upon bacterial cytochrome P450s and nitrophorins, and will utilize phage display to select mutants that bind the target compound in a defined position with respect to the heme. Our overall goal is to determine if the ligand binding sites of these different protein scaffolds can be induced to bind a variety of target ligands in a predetermined way.If so, then a general method for engineering novel catalysts with defined specificity may be possible. In addition, information about the inherent propensity of a particular protein architecture to accept a given ligand should provide insight into fundamentals of P450 substrate specificity.